Malate Dehydrogenase Collaborative CUREs Two Substrate Kinetics and Inhibition Module University of San Diego THE MALATE DEHYDROGENASE LABORATORIES Laboratory Page Overview of the Enzyme Kinetics Block of Laboratories 1 Introduction to the Study of Enzyme Kinetics and Enzyme Mechanisms 2 Review of the Roles of Malate Dehydrogenase 5 EXPERIMENT 1 Determination of the basic kinetic parameters, Km and Vmax 7 Does the Enzyme Obey Simple Michaelis Menten Kinetics? Introduction to the Design of the Experiment 7 Determination of Km for Oxaloacetate 9 Set Up of the Experiment you will perform 11 Determination of Km for NADH 13 Characterization of the Kinetic and Binding Properties of the Enzyme 15 EXPERIMENT 2 Determine the True Kinetic Parameters for 3 Phosphoglycerate Dehydrogenase at 16 pH 7.5 Set Up of the Experiment you will perform 21 Quantitative Analysis of Data 24 Group Project 27 Problem Set 1.1 27 EXPERIMENT 3 Inhibitors Competitive Inhibitors 29 Experiments to Investigate the Behavior of Structural Analogs and Competitive 31 Inhibitors on the Activity of Malate Dehydrogenase Set Up of the Experiment you will perform 31 Mixed Inhibitors 35 Set Up of the Experiment you will perform 36 Quantitative Analysis of Data 40 Problem Set 2.1 41 EXPERIMENT 4 Determination of Thermodynamic Parameters from Kinetic Experiments 41 Set Up of the Experiment you will perform 41 Problem Set 3.1 42 Overview of the Enzyme Kinetics Block of Laboratories In previous course work, the activity of an enzyme was used to follow the purification of the enzyme and to quantitate the amount of an enzyme present in a solution. In CHEM331, you have also been exposed to “one substrate” kinetics where you can determine the Km and Vmax from a single set of experiments by varying the concentration of the substrate. The experiments in this course focus on using measurements of the rate of enzyme activity to characterize the interaction of the substrates and inhibitory ligands with the enzyme, and to ascertain the overall rate limiting step in the enzyme catalyzed pathway for enzymes with two substrates. The first experiment will illustrate how the basic kinetic parameters, Km and Vmax, for an enzyme are determined and how careful analysis of the data obtained in such experiments can be used to determine whether Jessica & Ellis Bell Copyright 1 Malate Dehydrogenase Collaborative CUREs Two Substrate Kinetics and Inhibition Module University of San Diego and enzyme obeys "normal" behavior or might be subject to some type of "homotropic" regulation, where the activity is disproportionate to the substrate concentration and the activity of the enzyme is "regulated" in some way by changing substrate concentrations. As an important corollary to the actual experiments, the data analysis discussed in this chapter, both for the actual experiments and for the problem sets, illustrates how replicate measurements and appropriate data analysis can be used to determine whether the assumed equation used for the analysis is appropriate and you can maximize the information obtainable from your data. Introduction There are many different types of enzymes, categorized using the so-called "Enzyme Commission,” E.C., numbers into classes based upon the types of chemistry that they utilize in their reactions. What is it about enzymes that make them so attractive for study? Enzymatic activities play crucial roles in every type of life process in addition to being increasingly important in both drug design and synthesis and biotechnology. This is easily seen by considering the types of enzymes that might be found in a typical cell. Some enzymes are involved in the basic biochemistry and molecular biology of virtually every cell, whether they are associated with metabolism [the enzyme Glyceraldehyde-3-Phosphate Dehydrogenase is an excellent example: virtually all cells have a constant level of expression of the gene for Glyceraldehyde-3-Phosphate Dehydrogenase as it is involved in a critical step in glycolysis, common to virtually all cells], or the machinery of RNA transcription and protein synthesis: such enzymes are often referred to as “house-keeping” enzymes. Particular enzymes are sometimes associated only with specific tissues or cell types, or only with certain subcellular organelles and can be used as "marker enzymes" for that tissue or organelle [for example the Heart Isoenzyme of Lactate Dehydrogenase: LDH-H, is found only in aerobic muscle such as the heart rather than skeletal muscle which is designed to operate under anaerobic conditions: measurement of the amount of H-LDH can be used to determine whether a person has had a heart attack.] Similarly, the various Glycosyl transferases used in glycoprotein biosynthesis are found only in the Golgi membrane and their activity can be used as a marker for that subcellular organelle. Table.1 summarizes several tissue or organelle specific enzymes the measurement of whose activity has been useful in defining the tissue or organelle. Marker Enzymes: Membrane or Location Marker Enzyme Mitochondrial: Inner Membrane Succinate-Cytochrome c Reductase Rotenone Sensitive NADH Cytochrome c Oxidase Mitochondrial: Outer Membrane MonoAmine Oxidase Rotenone Insensitive NADH Cytochrome c Oxidase Endoplasmic Reticulum RNA Protein Synthesis Enzymes NADPH Cytochrome c Reductase Plasma Membrane 5' Nucleotidase Lectin Binding Oxytocin [or Hormone] Binding Golgi Apparatus Glycosyl Transferases Mitochondrial Matrix Glutamate Dehydrogenase Jessica & Ellis Bell Copyright 2 Malate Dehydrogenase Collaborative CUREs Two Substrate Kinetics and Inhibition Module University of San Diego Cytosol Glyceraldehyde 3 Phosphate Dehydrogenase Enzymes in both these first two categories are not only necessary for the everyday functions of a particular cell but often have their activity regulated to allow the cell to respond to normal changes in its environment. Virtually all cell types in eukaryotic biology differentiate during their life time and usually certain enzyme activities can be associated with the stage of differentiation of the cell: for example chondrocytes: [cells that help create bone formation] differentiate to produce specific types of collagen and the enzyme alkaline phosphatase [which is directly involved in releasing the phosphate that will be used in hydroxyapatite-the main mineral in bone or teeth formation]. The quantitation of the activity of alkaline phosphatase can be used to follow the level of differentiation of these cells. Finally, the activity of some enzymes indicates the presence of an invading organism. During the early stages of the AIDS crisis scientists measured the activity of the enzyme reverse transcriptase in infected tissue to show that a retrovirus was involved in the infection. Enzymes specific for an invading organism are often targets for drug design: again the AIDS crisis offers many examples. The reverse transcriptase and the HIV protease have both been targets of intensive research in recent years to find specific inhibitors to block their activity. Pepsin [left] and the HIV Protease [right] are both Aspartyl proteases. The catalytic aspartate groups are shown on the ribbon diagram of pepsin, which is a monomeric enzyme, and as can be seen are similarly located in the HIV protease although each group is contributed by a different subunit. The two subunits of the HIV protease are identical and together the dimer shows overall structural similarity to pepsin. So, what type of questions do we want to explore with enzymes? Any enzyme catalyzed reaction can be broken down into three simple phases, see scheme 1: 1] substrate binding, 2] chemical catalysis, and 3] product release. To understand an enzyme, we must be able to examine each of these phases and document their individual contributions to the overall activity of the enzyme. In the first phase, we want to know what is the order of addition of substrates if there are more than one substrate, and how tightly do the substrates bind. In the second phase, we want to be able to ask Jessica & Ellis Bell Copyright 3 Malate Dehydrogenase Collaborative CUREs Two Substrate Kinetics and Inhibition Module University of San Diego Eukaryotic Glutamate Dehydrogenases, the structure of bovine glutamate dehydrogenase is shown on the left, are multisubunit enzymes which show complex allosteric behavior involving both substrates [homotropic regulation] and a variety of other ligands [heterotropic regulation] such as ADP, GTP, Leucine, Zinc and a wide variety of other ligands. Enzyme kinetic studies have played an important role in elucidating how substrates and regulatory ligands interact with this complex enzyme. The enzyme has six identical subunits [each subunit is shown in a different color] with complex subunit interactions mediated by the "antennae" [top and bottom] which connect three subunits within each half of the enzyme questions about the nature of the overall rate limiting step of the reaction. In the third phase, like the first phase, the order of product release is important. Since an enzyme is often present with a mixture of both substrates and products, we may also want to know whether complexes where, for example, one substrate and one product can bind simultaneously to the enzyme exit. Such complexes may well play important roles in the regulation of the activity of the enzyme if they are more stable than the "normal" enzyme-substrate or enzyme-product complexes. In the first two classes of enzymes considered above (housekeeping, cell type/organelle specific), in addition